The traditional processing of coca beans to generate cocoa flavor requires two steps—a fermentation step, which includes air-drying of the fermented material, and a roasting step.
During fermentation the pulp surrounding the beans is degraded by micro-organisms and the sugars contained in the pulp are mainly transformed to acids. In the course of the fermentative process these acids slowly diffuse into the bean eventually causing an acidification of the cellular material. Furthermore, during fermentation peptides of different sizes are generated as well as high levels of hydrophobic free amino acids, which are mainly attributed to the activity of specific proteinases. This specific mixture of peptides and hydrophobic amino acids is thought to be the cocoa-specific flavor precursors.
Research to date has focused on the different proteolytic enzymes involved in these reactions. A number of different types of enzymes, such as an aspartic endoproteinase, a cysteine endoproteinase or a carboxypeptidase have been found to participate in these degradative reactions leading to the formation of the cocoa flavor peptide/amino acid precursor pool.
During the second step of cocoa flavor production—the roasting step—the oligopeptides and amino acids generated during the fermentation stage are subjected to a Maillard reaction in the presence of reducing sugars in the mixture, yielding substances thought to be responsible for the typical cocoa flavor.
There have been attempts to artificially produce cocoa flavor in the past, such as, by subjecting acetone dried powder prepared from unfermented ripe cocoa beans to autolysis at a pH of 5.2 followed by roasting in the presence of reducing sugars. It was taught that under these conditions preferentially free hydrophobic amino acids and hydrophilic peptides would be generated. The peptide pattern obtained from this process was found to be similar to that of extracts from fermented cocoa beans.
Analysis of free amino acids revealed that Leu, Ala, Phe and Val were the predominant amino acids liberated in fermented beans or autolysis (Voigt et al., Food Chem. 49 (1994), 173–180). In contrast to these findings no cocoa-specific flavor could be detected when the above powder was subjected to autolysis at a pH of 3.5. Few free amino acids were found in the by product of the autolysis, but there were a large number of hydrophobic peptides formed.
Synthetic mixture of free amino acids whose composition resembles that found in fermented beans also have been found to not produce the cocoa flavor desired. These findings indicate that both the peptides and the amino acids are important in producing cocoa flavor (Voigt et al., Food Chem. 49 (1994), 173–180.
To date, little attention has been paid to the protein pool from which the peptide/amino acid flavor precursor pool is generated from, since cocoa proteins are often difficult to isolate. One of the major reasons is because that coca seeds contain a high amount of polyphenols and fat. Separating the polyphenols and fat traditionally requires the use of lipophilic organic liquids, such as acetone. The use of these liquids often result in the removal of lipophilic flavor precursors and active substances. Another reason is because of the poor solubilization of proteins purified with acetone, resulting in a poor recovery of the total proteins.
To date, four major proteins with an apparent molecular weight of 14.5, 21, 31 and 47 kDa, have been identified before fermentation in cocoa bens. These proteins are thought to give rise to the peptide/amino acid pool responsible for producing the cocoa flavor.
It is an object of the present invention to further elucidate and identify other protein responsible for producing the cocoa flavor in sufficient detail to eventually provide means for improving the preparation of cocoa flavor and cocoa flavor substitutes.